(a) Field of the Invention
The invention relates to novel bovine and porcine genomic sequences for the SRY gene, a method for genetic sex determination of bovine or porcine tissue, and a method for the genetic selection of sexual phenotype in domestic animals.
(b) Description of Prior Art
In mammals, males are heterogametic (XY) while females are homogametic (XX), and the male phenotype is correlated with the presence of the Y chromosome. It has been postulated that a control gene, the testes determining factor, is present on the Y chromosome and acts as a developmental switch between the sexes. In the presence of the testes determining factor (i.e. XY), indifferent primitive gonadal tissue develops into testes with a resulting male body phenotype. In the absence of the testes determining factor (i.e. XX), indifferent primitive gonadal tissue fails to receive the testes determining signal, and develops into ovaries with a resulting female body phenotype. The concordance between chromosomal sex and phenotypic sex is not absolute, and the rare exceptions to the rule have been valuable in defining the molecular characteristics of TDF. In 1987, a gene specific to the Y chromosome termed zinc finger on the Y chromosome (ZFY) was isolated and proposed as a candidate gene for the testes determining factor and male sex determination (Page et al., 1987, Cell, 51:1091-1104). Further characterization of ZFY, particularly the facts that a highly homologous gene (ZFX) was found on the X chromosome and that the ZFY gene in marsupials is autosomal, led to the rejection of ZFY as the testes determining factor. In 1990, a new candidate gene for the testes determining factor, named Sex-Determining Region Y or SRY, was described in humans and mice (Sinclair AG et al., 1990, Nature, 346:240-244; Gubbay Jet al., 1990, Nature, 346:245-250). When the mouse SRY was introduced into XX mouse embryos via transgenic techniques, phenotypic male mice were generated (Koopman P et al., 1991, Nature, 351:117-121), and it is now generally accepted that SRY is the testes determining factor or genetic switch for the male phenotype in mammals. Structurally the SRY gene contains a centrally located region termed the "High Mobility Group" (HMG) box, which shows reasonably good sequence conservation between species. Interestingly, sequences on either side of the HMG box show a remarkable lack of sequence conservation even between closely related species (Whitfield LS et al., 1993, Nature, 364:713-715). Furthermore the SRY gene is represented uniquely on the Y chromosome, with no X-chromosome homologous sequences. These characteristics make the SRY gene an ideal target for gene based methods of sex determination and sexual phenotype selection.
Manipulation of sex ratios via early determination of sex of embryos is of potential commercial value to the cattle as well as other domesticated animal industries. Several methods have been described for sexing embryos, including X-linked enzyme assays, Y-linked HY antigen assays, cytogenetic assays and Y-linked DNA probes (VanVliet RA et al., 1989, Theriogenology, 32(3):421-438). Reliability, speed and acceptable rates of pregnancy are vital for commercial application of any method of embryo sexing. Recently a novel method for embryo sexing involving the in vitro amplification of Y-chromosome specific repetitive DNA sequences has been described (Peura T et al., 1991, Theriogenology, 35(3):547-555). This method is based on polymerase chain reaction (PCR) amplification technology, an enzymatic method to amplify target fragments of DNA in vitro (Saiki, R. K. et al., 1988, Science, 239:487-491)- Repetitive DNA sequences are repeated sequences of non-coding DNA found scattered throughout the genome. Their function is poorly understood. Repetitive sequences on the Y chromosome can be Y-chromosome specific or they can cross react with repetitive sequences on autosomes. Repetitive sequences can be associated with the Y chromosome but may not necessarily be associated with the male sexual phenotype since they do not code for genes involved with sexual differentiation, and since their highly repetitive nature increases the chances of translocation of sequences to the X chromosome or autosomes. Some Y chromosome repetitive sequences are not Y chromosome specific but are rather Y chromosome enhanced, and are found elsewhere in the genome. More recently, Y-chromosome specific single gene sequences have been used as targets for PCR based sexing regimes, both in lab animals (Kunieda T et al, 1992, Biology of Reproduction, 46:692-697) and domesticated animals (Kirkpatrick B. W. and Monson R. L., 1993., J. Reprod. Fertility, 98:335-340). Domesticated animal single gene targets for PCR based sexing methods have been the Zinc Finger Y chromosome gene, or ZFY (Page et al., 1987, Cell, 51:1091-1104), a gene which is highly conserved between domesticated species and which has an X-chromosome homologous gene, ZFX.
Georges et al. in Patent Cooperation Treaty Application Publication No. 90/15155 on Dec. 13, 1990, disclose a probe specific to Y chromosome based on Y specific repetitive sequences involving about 40 bp and approximately 100,000 copies per genome. This sequence is correlated with the Y chromosome but is not necessarily correlated with male sexual phenotype, due to its highly repetitive nature. This probe is more likely to give false positive results because of its high incidence per genome and thus may not be ideal for the sexing of bovine embryos.
Kwoh D. Y. et al. in U.S. Pat. No. 5,055,393 issued on Oct. 8, 1991, disclose the prenatal sex determination of bovine cells using male-specific oligonucleotides which are repeated male-specific sequences. Again, because these sequences are repeated, false positive results may be expected.
Reed K. et al. in Canadian Patent No. 1,296,770 issued on Feb. 25, 1992, disclose a method for the sex determination in ruminants using bovine repeated sequences on the Y chromosome BRY-1. The main disadvantage of this method is that it is based on repetitive sequence technology and not on gene sequences. Thus, it is not an absolute marker for male sexual determination, nor is it a means for phenotypic manipulation via genetic means.
Ellis S. et al. in Canadian Patent No. 1,306,431 issued on Aug. 18, 1992, disclose nucleic acid probes for prenatal sexing using male-specific repetitive sequences present in a range at about 10 to 5200 copies per genome. This method is not ideal, since it is based on repetitive, non coding sequences specific or enhanced on the Y chromosome.
Kudo T. et al. in European Patent Application Publication No. 546,762 on Jun., 16, 1993, disclose sexing methods for bovine embryos using repetitive sequences, some of which are Y-specific while others are gender non-specific. The main disadvantage of this method is that it is not based on gene sequences, but rather is based on repetitive sequences.
There is nothing in the prior art to indicate that any DNA segments exist in bovine or porcine male DNA which could be used to determine the sex of bovine or porcine tissue with no possibility of false-positive result.
There is nothing in the prior art to suggest that said DNA segments could be useful for in vivo manipulation of sexual phenotype in domestic animals.
It would be highly desirable to be provided with bovine or porcine genomic sequences which would be specific to bull or boar DNA but absent from cow or sow DNA and would not be repeated, repetitive or a so-called satellite DNA.
It would be highly desirable to be provided with a method for the genetic sex determination of bovine or porcine tissue.
It would be highly desirable to be provided with Y chromosome male specific gene sequences including coding sequences, promoter and control sequences useful for the in vivo manipulation of sexual phenotype in domestic animals.
It would be highly desirable to be provided with a method for introducing controllable gene sequences into the genome of domestic animals for the purpose of sexual phenotype, control and selection.